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Melanocytes in the Testes of Eupemphix nattereri Anura LeiuperidaeHistological Stereological and Ultrastructural Aspects.

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THE ANATOMICAL RECORD 290:795–800 (2007)
Melanocytes in the Testes of Eupemphix
nattereri (Anura, Leiuperidae):
Histological, Stereological, and
Ultrastructural Aspects
São Paulo State University, Department of Biology, Institute of Biosciences,
Literature and Exact Sciences, São José do Rio Preto, São Paulo, Brazil
Ectothermic vertebrates have a well-developed system of melanincontaining cells, which localize in several organs and tissues and compose
an extracutaneous pigmentary system. This research aimed at characterizing histological and ultrastructural patterns of pigmented cells in the
testes of the anura Eupemphix nattereri (Steindachner, 1963), including
the stereological and quantitative evaluation of this cell type in the
gonads. Ten adult males were collected in Nova Itapirema, São Paulo,
Brazil, and submitted to morphological studies with light and transmission electron microscopy. The testis presents a great number of large cells
with many brown granules and long cytoplasmic processes. The pigmented cells found in the testis are structurally similar to melanocytes,
characterized by large amounts of melanosomes. The cells may be in intimate contact with the same cell type, with myoid cells surrounded by a
large amount of collagen fibers, Leydig cells, and next to fibroblasts. The
distribution and amount of extracutaneous melanocytes is variable when
other organs and membranes are analyzed, allowing the establishment of
species-specific patterns for the extracutaneous pigmentary system. Anat
Rec, 290:795–800, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: melanocyte; melanosomes; extracutaneous pigmentary system; testes; anura
Ectothermic vertebrates have a well-developed system
of melanin-containing cells, distributed in several organs
(liver, spleen, lung, heart, thymus, and gonads) and tissues (meninges and connective tissue surrounding blood
vessels) and composing an extracutaneous pigmentary
system (Gallone et al., 2002).
Melanocyte-like cells, which derive from the neural
crest (Sichel et al., 1997) and produce and store melanin
inside spherical or ovoid structures called melanosomes
(Agius and Roberts, 2003), have been observed in the epidermis and in several organs (Agius and Agbede, 1984;
Zuasti et al., 1998; Oliveira and Zieri, 2005). These cells
are large and irregular in shape, with a great amount of
intensely pigmented cytoplasm, and may be seen under
stereoscope microscopy. They can have star-like or punctiform morphology, due to the presence or absence of
cytoplasmic processes, respectively (Oliveira and Zieri,
Melanomacrophage centers are usually found in hematopoietic organs of basal vertebrates (Agius, 1980;
Agius and Agbede, 1984) and present different types of
granules inside the cytoplasm, which can indicate differGrant sponsor: Coordenação de Aperfeiçoamento de Pessoal
de Nı́vel Superior (CAPES); Grant sponsor: São Paulo State
Research Foundation (FAPESP); Grant numbers: 02/08016-9
and 05/02919-5; Grant sponsor: The National Council of Scientific and Technological Development; Grant number: 301111/05-7.
*Correspondence to: Classius de Oliveira, São Paulo State
University (UNESP), Department of Biology, Rua Cristóvão
Colombo, 2265, Jardim Nazareth, CEP: 15054 000, São José do
Rio Preto, São Paulo, Brazil. E-mail:
Received 21 February 2006; Accepted 22 March 2007
DOI 10.1002/ar.20553
Published online 31 May 2007 in Wiley InterScience (www.
ent chemical substances, such as melanin, hemosiderin,
and lipofuscin, resulting from the endosomal–lysosomal
digesting processes (Agius and Agbede, 1984; Herraez
and Zapata, 1991).
In amphibians, pigmented cells appear in tegument
and in lung as typical melanocytes and in spleen and
liver as melanomacrophage centers (Zuasti et al., 1998).
Melanomacrophages found in the liver of amphibians
are also known as Kupffer cells (Sichel et al., 1997;
Zuasti et al., 1998; Prelovsek and Bulog, 2003).
Melanocytes are characteristically distributed in
organs and tissues, with species-specific patterns of
occurrence, types, and amounts (Aoki et al., 1969; Geremia et al., 1984; Gopalakrishnakone, 1986; Cicero et al.,
1989; Pederzoli and Trevisan, 1990; Trevisan et al.,
1991; Zagal’skaia, 1994; Christiansen et al., 1996; Sichel
et al., 1997; Akulenko, 1998; Zuasti et al., 1998; Rund
et al., 1998; Barni et al., 1999, 2002; Johnson et al.,
1999; Oliveira et al., 2002, 2003; Prelovisek and Bulog,
2003; Oliveira and Zieri, 2005). It has thus been proposed that patterns of extracutaneous pigmentary systems may be established for each species (Oliveira and
Zieri, 2005).
Previous studies have described large amounts of melanocyte-like pigmented cells in the interstitial testicular
tissue in Physalaemus fuscomaculatus (Aoki, 1969),
Physalaemus cuvieri (Oliveira et al., 2002, 2003), and
Eupemphix nattereri (Oliveira and Zieri, 2005), presenting a brief and gross morphologic description of these
cell types. Further characterization of the cells would be
of great interest. Testicular pigmentation has been used
as an important phenotypical character in the taxonomy
and phylogeny of Dendrobatidae (Grant et al., 2006).
A few recent studies have described the extracutaneous pigmentary system of anuran amphibians (P. cuvieri,
Oliveira et al., 2002, 2003; P. fuscomaculatus, Aoki
et al., 1969; E. nattereri, Oliveira and Zieri, 2005; Rana
esculenta, Cicero et al., 1989; Sichel et al., 1997; Barni
et al., 1999, 2002; Gallone et al., 2002; Rana ridibunda,
Akulenko, 1998; Xenopus laevis, Sichel et al., 1997; Zuasti
et al., 1998). The present study aimed at characterizing
histological and ultrastructural patterns of pigmented
cells in the testis of the anuran Eupemphix nattereri,
including stereological and quantitative evaluation of this
cell type in the gonads.
Ten adult male Eupemphix nattereri (Anura, Leiuperidae) were collected at temporary ponds (218040 4000 S;
498320 2300 W) in Nova Itapirema, São Paulo, Brazil,
between September and October of 2003, when they
were in the reproductive activity period. In the laboratory, the individuals were anesthetized, killed, and submitted to morphological studies. Animal handling and
experiments were done according to the ethical guidelines of the São Paulo State University (Unesp), following the Guide for Care and Use of Laboratory Animals.
All animals were weighed and dissected out by medium incision exposing the reproductive organs. After
this procedure, the testes were removed and weighted,
and immediately immersed in Karnovsky fixative solution (0.1 M Sörensën phosphate buffer pH 7.2 containing
5% paraformaldehyde and 2.5% glutaraldehyde). The
material was dehydrated in an alcohol gradient and embedded in historesin (Leica-historesin embedding kit).
Sections of 2 mm were stained with hematoxylin–eosin
and observed under an Olympus BX60 microscope.
Samples for transmission electron microscopy observations were fixed for 2 hr at 258C in 3% glutaraldehyde
and 0.25% tannic acid at pH 7.3 in Millonig’s buffer. After washing in the same buffer, the samples were postfixed for 1 hr in 1% osmium tetroxide diluted in same
buffer, dehydrated in acetone, and embedded in araldite
resin (Cotta-Pereira et al., 1976). Ultrathin silver sections (75 nm) were obtained using a diamond knife and
stained with 2% uranyl acetate for 20 minutes (Watson,
1958) and 2% lead citrate in 1 N sodium hydroxide solution (Venable and Coggeshall, 1965) for 8 min, and
examined under a Leo-Zeiss EM - 906 transmission electron microscope operating at 80 kV.
Stereology Assays
Ten histological fields for each animal were examined
using an image analyzing system software (Image ProPlus, Media-Cybernetics, Inc.). Stereological analyses
were performed with the Weibel’s multipurpose graticulate with 120 points and 60 test lines (Weibel, 1978), to
compare the relative proportion (relative volume) of pigmented cells. The absolute volume of these cells was
determined by multiplying the relative volume values by
the mean testes weight, based on the determination that
1 mg of fresh tissue has a volume of approximately
1 mm3, according to Vilamaior et al. (2006).
To correlate the testicular melanocytes and the
remaining variables (body weight, testes weight, percentage of interstitial cells, germinative cells, and other
cells types in the testis), outliers were identified and the
Shapiro–Wilk test was used to test for normality. The
Pearson correlation coefficient for parametric data was
used for analysis of the results.
Eupemphix nattereri have paired and ovoid testes,
measuring around 4.82 6 0.78 mm length, obtained
from right testes from 10 animals. They present marked
pigmentation in the testicular capsule (Fig. 1A,B) and
around the seminiferous tubules, intimately associated
with interstitial cells (Fig. 1C,D) so that the gonads
present a dark brown color.
This intense pigmentation is due to the presence, in
the testes, of great numbers of large cells with many
brown granules and long cytoplasmic processes distributed in both interstitial and capsular regions of the
gonads (Fig. 1C,D). Cell borders could not be identified
by histological examination, because the cells displayed
a very high variation of irregular shapes.
Evaluation of the pigmented cells under transmission
electron microscopy showed large irregular nuclei, conspicuous nucleoli, and condensed chromatin in the
peripheral region (Fig. 2A). The cytoplasm showed
numerous processes, with poorly developed organelles,
and consisted mostly of electron-dense corpuscles containing melanin (Fig. 2B). The cytoplasmic region pigment
granule-free present a few Golgi’s cisternae (Fig. 2D,E),
and in association with these pigment granules, a prominent cytoskeleton can be observed (Fig. 2C). Melanosomes
Fig. 2. Transmission electron microscopy of E. nattereri melanocytes. A,B: Melanocyte shows mature granules, with the cytoplasm
full of melanosomes of different shapes, sizes, and electron densities.
C: Detail of the melanocyte’s cytoplasm showing cytoskeleton elements: microtubules (large arrows) and actin filaments (asterisk). D,E:
Detail of melanocyte’s cytoplasm demonstrating Golgi complex (GC)
in association with secreting vesicles (small arrows). F: Cytoplasmic
process of a melanocyte with melanosomes presenting different sizes
and development stage (arrows). Mi, mitochondria; N, nucleus; Nu,
nucleolus; PG, pigment granules.
Fig. 1. A,B: Anatomical (A) and histological (B) general view of the
testis of Eupemphix nattereri, showing intense pigmentation testicular.
C: Pigmentation of the testicular capsule (arrows) and around the
seminiferous tubules (asterisks). D: Detail of melanocytes in the testicular interstitium. fb, fatty body; t, testis; k, kidney.
presented round or ovoid shape with variable size and
degree of melanization (Fig. 2F). The main characteristic
of the mature pigmented cell in the testis evaluated in
the present study is their homogeneous appearance and
the presence of well-defined melanin granules (Fig. 2B).
The pigmented cells and their processes may be in
intimate contact and/or high proximity with fibroblasts
(Fig. 3A–C), with myoid cells surrounded by a large
amount of collagen fibers (Fig. 3E,F), Leydig cells (not
documented), and next to the same cell type, constituting a homotypic cell contact (Fig. 3D). Fibroblast and
myoid cells were identified by ultrastructural analysis,
using the evaluation of the basal membrane in the
myoid cells and absence of this structure in the fibroblast (Fig. 3F). Close contact, similar to focal adhesion,
was also occasionally observed between fibroblasts, next
to the pigmented cells (Fig. 3B).
Pigmented cells found in the testes are structurally
similar to melanocytes, characterized by a large amount
of melanin pigments in the interior of electron-dense corpuscles containing melanin, the melanosomes. The absence of the protein synthesis machinery organelles,
such as granular endoplasmic reticulum, polysomes, and
lysosomal-like vesicles or multivesicle bodies, typical of
macrophages, were used here as evidence that this
cell type is not a component of the melanomacrophage
Stereological analyses confirmed that most of the area
in the testis (58.06 6 9.53%) and the absolute volume
(8.12 6 1.33 mg) are occupied by germ cells and that
melanocytes occupy 5.08 6 2.41% of the total area and
0.71 6 0.33 mg of absolute volume. The melanocytes’
absolute and relative volumes were not significantly correlated to body or testicular weight (Pearson correlation,
TABLE 1. Quantitative Exploratory Analysis of the
Testicular Components in Eupemphix nattereria
Biometrical data
Body weight (g)
Testes weight (g)
Stereological data
Relative volume (%)
Germ cells
Other components
Absolute volume (mm3)
Germ cells
Other components
Animal (n ¼ 10); histological fields (n ¼ 10). Values represent mean 6 SD.
Fig. 3. Transmission electron microscopy of Eupemphix nattereri
melanocytes. All figures demonstrate the association of this cell type
with other cells, indicative of the cell communication. A–C: Proximity
to fibroblast (F) processes (dark arrows). D: Spot junction with plasmatic membrane of another melanocyte, characterizing a homotypical
cell interaction (white arrow). E,F: Melanocytes closely associated
to fibroblasts (F), with collagen fibers around (Co) and myoid cells
(MC) with the basal membrane (thin arrows). N, nucleus; PG, pigment
P 0.05), or to the volumetric values of interstitial and
germinative cells. In addition, melanocytes volume
showed negative correlation with the other testicular
components (Pearson correlation, r ¼ 0.71; P ¼ 0.01;
Table 1).
Ectothermic animals have cells with pigmented cytoplasm, seen in different organs, which form the extracutaneous pigmentary system whose function is still poorly
understood (Zuasti et al., 1998). The pigmented cells
have different names, such as melanophores, melanocytes, melanomacrophages, melanin-containing mononuclear phagocytes, and melanophages. In amphibians
(Sichel et al., 1997, Zuasti et al., 1998, Prelovisek and
Bulog, 2003) and reptiles (Christiansen et al., 1996;
Rund et al., 1998; Johnson et al., 1999), similar cell
types present in the liver are called Kupffer cells.
These cells are morphologically similar to melanocytes
(Agius, 1980; Zuasti et al., 1990, 1998) and macro-
phages. In ectothermic animals, macrophages have large
amounts of melanin in the cytoplasm and are thus called
melanomacrophages (Agius, 1980; Agius and Agbede,
1984; Macchi et al., 1992; Micale and Patti, 1993; Messeguer et al., 1994). Melanocytes originate from the neural
crest, whereas melanomacrophages derive from hematopoietic stem cells (Sichel et al., 1997). According to the
morphologic characteristics of the cells analyzed in
testes and associated membranes in the present study,
they were referred to as melanocytes.
The strong testicular pigmentation observed in E. nattereri is evidenced by cell types that are ultrastructurally similar to melanocytes. These cells are present in
the skin (Trevisan et al., 1991) and in several visceras
(Geremia et al., 1984), as well as in the testicular capsule and in close contact with interstitial cells, as
described in P. fuscomaculatus (Aoki et al., 1969), Bombina bombina (Gollmann et al., 1993), P. cuvieri (Oliveira
et al., 2002, 2003), and E. nattereri (Oliveira and Zieri,
Melanocytes are large irregular cells, with poorly
developed organelles, a great number of melanosomes,
and long dendritic processes with abundance of cytoskeleton, such as microtubules and actin filaments (Aoki
et al., 1969). In E. nattereri testes, melanocytes have
melanosomes of different sizes and degrees of melanization, suggesting the occurrence of melanogenic activity
as described by Sichel et al. (1997) and Gallone et al.
(2002) in Rana esculenta. The melanogenesis in E. nattereri testes is suggested here due the presence of the
several Golgi’s cisterns in association with secreting
vesicles. But the confirmation of this process will certainly be a new focus of further investigations in our
research group.
Melanosynthesis has also been observed in Kupffer
cells in amphibian liver (Cicero et al., 1989; Sichel et al.,
1997), as shown by the presence of premelanosomes in
several stages of differentiation and by the activity of
dopa-oxidase in R. esculenta (Gallone et al., 2002) and
Proteus anguinos (Prelovsek and Bulog, 2003). Melanosynthesis, however, has not been observed in melanomacrophages of some species of fish, showing that the melanin
inside the cells derives from phagocytosis of melanosomes originating from melanocytes (Agius and Agbede,
1984; Tsujii and Seno, 1990; Zuasti et al., 1990; Herraez
and Zapata, 1991).
In teleosts, Besseau and Faliex (1994) and Grier and
Taylor (1998) have described the presence of melanomacrophages in testicular structures and that they might
be involved with the gonadal regression observed after
maturation of germinative cells. In E. nattereri, melanomacrophages have not been observed in the testicular
interstitium. The cells could, however, be present in low
density or only in specific phases of the reproductive
cycle or yet in response to infectious processes, when
they could then be identified. In anurans and teleosts,
these cells are clearly evident in hematopoietic organs
such as the liver and spleen (Cicero et al., 1989; Zuasti
et al., 1990; Sichel et al., 1997) and in the kidneys
(Zuasti et al., 1989).
Functions of the melanin inside melanomacrophages
include a possible role in the absorption and neutralization of free radicals, cations, and other potentially toxic
agents derived from the degradation of phagocytosed
cells (Zuasti et al., 1989), as well as in the thermoregulation process in reptiles (Golapakrishnakone, 1986).
Barni et al. (1999) reported an increase of melanic cells
in the liver of R. esculenta during winter. In E. nattereri
the function of these pigmented cells yet cannot be
completely explained, but the evidence of the thermoregulation role may be indicated here. However, this hypothesis will be tested in further experimental assays.
The cells are structurally very similar when compared
in other species such as B. bombina (Gollmann et al.,
1993) and P. cuvieri (Oliveira et al., 2002, 2003). However, the distribution and amount of extracutaneous melanocytes is variable when other organs and membranes
are analyzed, allowing the establishment of species-specific patterns for the extracutaneous pigmentary system
(Oliveira and Zieri, 2005).
The present study describes characteristics that can
be further explored in the characterization of anurans,
providing morphological results of importance for the
use of other approaches involving physiological and
phylogenetic aspects. The anuran extracutaneous pigmentary system has been a target of research in our laboratory with emphasis on reproductive organs, and other
complementary immunocytochemical and enzymological
methodologies have been established. In this sense,
major information about the physiology of these cells
will be incorporated to the literature in future.
This study is part of Master’s thesis presented by R.Z.
to the Animal Biology Post-graduation Course, Ibilce/
Unesp. The authors thank Luiz Roberto Falleiros Júnior
and Rosana Silistino de Souza for technical assistance.
R.Z. received a fellowship from the Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior. S.R.T.
thanks the National Council of Scientific and Technological Development.
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ultrastructure, aspects, tester, stereological, melanocytic, nattereri, anura, eupemphix, leiuperidaehistological
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